Isolated, tyrosinase derived peptides and uses thereof

ABSTRACT

The invention relates to the identification of complexes of human leukocyte antigen molecules and tyrosinase derived peptides on the surfaces of abnormal cells. The therapeutic and diagnostic ramifications of this observation are the subject of the invention.

RELATED APPLICATION

This application is a continuation-in-part of Ser. No. 08/203,054 filedon Feb. 28, 1994, which is a continuation-in-part of application Ser.No. 08/081,673, filed Jun. 23, 1993, which is a continuation in part ofU.S. patent application Ser. No. 054,714, filed Apr. 28, 1993 which is acontinuation-in-part of U.S. patent application Ser. No. 994,928, filedDec. 22, 1992, now abandoned.

FIELD OF THE INVENTION

This invention relates to isolated peptides, derived from tyrosinasewhich are presented by HLA-A2 and HLA-B44 molecules and uses thereof. Inaddition, it relates to the ability to identify those individualsdiagnosed with conditions characterized by cellular abnormalities whoseabnormal cells present complexes of these peptides and HAL-A2 andHAL-B44, the presented peptides, and the ramifications thereof.

BACKGROUND AND PRIOR ART

The process by which the mammalian immune system recognizes and reactsto foreign or alien materials is a complex one. An important facet ofthe system is the T cell response. This response requires that T cellsrecognize and interact with complexes of cell surface molecules,referred to as human leukocyte antigens ("HLA"), or majorhistocompatibility complexes ("MHCs"), and peptides. The peptides arederived from larger molecules which are processed by the cells whichalso present the HLA/MHC molecule. See in this regard Male et al.,Advanced Immunology (J. P. Lipincott Company, 1987), especially chapters6-10. The interaction of T cell and complexes of HLA/peptide isrestricted, requiring a T cell specific for a particular combination ofan HLA molecule and a peptide. If a specific T cell is not present,there is no T cell response even if its partner complex is present.Similarly, there is no response if the specific complex is absent, butthe T cell is present. This mechanism is involved in the immune system'sresponse to foreign materials, in autoimmune pathologies, and inresponses to cellular abnormalities. Recently, much work has focused onthe mechanisms by which proteins are processed into the HLA bindingpeptides. See, in this regard, Barinaga, Science 257: 880 (1992);Fremont et al., Science 257: 919 (1992); Matsumura et al., Science 257:927 (1992); Latron et al., Science 257: 964 (1992).

The mechanism by which T cells recognize cellular abnormalities has alsobeen implicated in cancer. For example, in PCT applicationPCT/US92/04354, filed May 22, 1992, published on Nov. 26, 1992, andincorporated by reference, a family of genes is disclosed, which areprocessed into peptides which, in turn, are expressed on cell surfaces,which can lead to lysis of the tumor cells by specific CTLs. The genesare said to code for "tumor rejection antigen precursors" or "TRAP"molecules, and the peptides derived therefrom are referred to as "tumorrejection antigens" or "TRAs". See Traversari et al., Immunogenetics 35:145 (1992); van der Bruggen et al., Science 254: 1643 (1991), forfurther information on this family of genes.

In U.S. patent application Ser. No. 938,334, the disclosure of which isincorporated by reference, nonapeptides are taught which bind to theHLA-A1 molecule. The reference teaches that given the known specificityof particular peptides for particular HLA molecules, one should expect aparticular peptide to bind one HLA molecule, but not others. This isimportant, because different individuals possess different HLAphenotypes. As a result, while identification of a particular peptide asbeing a partner for a specific HLA molecule has diagnostic andtherapeutic ramifications, these are only relevant for individuals withthat particular HLA phenotype. There is a need for further work in thearea, because cellular abnormalities are not restricted to oneparticular HLA phenotype, and targeted therapy requires some knowledgeof the phenotype of the abnormal cells at issue.

The enzyme tyrosinase catalyzes the reaction converting tyrosine todehydroxyphenylalanine or "DOPA" and appears to be expressed selectivelyin melanocytes (Muller et al., EMBOJ 7: 2715 (1988)). An early report ofcDNA for the human enzyme is found in Kwon, U.S. Pat. No. 4,898,814. Alater report by Bouchard et al., J. Exp. Med. 169: 2029 (1989) presentsa slightly different sequence. A great deal of effort has gone intoidentifying inhibitors for this enzyme, as it has been implicated inpigmentation diseases. Some examples of this literature include Jinbow,WO9116302; Mishima et al., U.S. Pat. No. 5,077,059, and Nazzaropor, U.S.Pat. No. 4,818,768. The artisan will be familiar with other referenceswhich teach similar materials.

U.S. patent application Ser. No. 08/081,673, filed Jun. 23, 1993 andincorporated by reference, teaches that tyrosinase may be treated in amanner similar to a foreign antigen or a TRAP molecule--i.e., it wasfound that in certain cellular abnormalities, such as melanoma,tyrosinase is processed and a peptide derived therefrom forms a complexwith HLA molecules on certain abnormal cells. These complexes were foundto be recognized by cytolytic T cells ("CTLs"), which then lyse thepresenting cells. The ramifications of this surprising and unexpectedphenomenon were discussed. Additional peptides have now been found whichalso act as tumor rejection antigens presented by HLA-A2 molecules.These are described in Ser. No. 08/203,054, filed Feb. 28, 1994 andincorporated by reference.

It has now been found that additional peptides derived from tyrosinaseare tumor rejection antigens in that they are presented by MHC moleculeHLA-B44, and are lysed by cytolytic T cells.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 describes, collectively, cell lysis studies. In particular:

FIG. 1A shows lysis of cell line LB24-MEL, by CTL 210/9;

FIG. 1A' shows lysis of cell lines LB24-MEL, by CTL IVSB;

FIG. 1B shows lysis of cell line SK29-MEL, by CTL 210/9;

FIG. 1B' shows lysis of cell line SK29-MEL, by CTL IVSB;

FIG. 1C shows lysis of cell line LB4.MEL, by CTL 210/9;

FIG. 1C' shows lysis of cell line LB4.MEL, by CTL IVSB;

FIG. 1D shows lysis of cell line SK23.MEL, by CTL 210/9;

FIG. 1D' shows lysis of cell line SK23.MEL, by CTL IVSB;

FIG. 1E shows lysis of cell line LE516.MEL, by CTL 210/9;

FIG. 1E' shows lysis of cell line LE516.MEL, by CTL IVSB;

FIG. 1F shows lysis of cell line SK29-MEL.1.22 which has lost HLA-12expression, by CTL 210/9;

FIG. 1F' shows lysis of cell line SK29-MEL.1.22 which has lost HLA-A2expression, by CTL IVSB;

FIG. 1G shows lack of lysis of MZ2-MEL, by CTL 210/9;

FIG. 1G' shows lack of lysis of MZ2-MEL, by CTL IVSB;

FIG. 1H shows lysis of the loss variant in FIGS. 1F and 1F' aftertransfection with a gene for HLA-A2, by CTL 210/9;

FIG. 1H' shows lysis of the loss variant in FIGS. 1F and 1F' aftertransfection with a gene for HLA-A2, by CTL IVSB;

FIG. 1I shows lysis of the loss variant in FIGS. 1F and 1F' aftertransfections with a gene for HLA-A2, by CTL 210/9;

FIG. 1I' shows lysis of the loss variant in FIGS. 1F and 1F' aftertransfections with a gene for HLA-A2, by CTL IVSB.

FIG. 2 presents studies of TNF release of CTL IVSB.

This cell line is also referred to as SK29-CTL IVSB in the disclosurewhich follows. CTL IVSB and SK 29-CTL IVSB are used interchangeably.

FIG. 3 depicts studies of TNF release of CTL 210/9.

This cell line also referred to as LB 24-CTL 210/9 in the disclosurewhich follows. CTL 210/9 and LB24-CTL 210/9 are used interchangeably.

FIGS. 4A and 4B depict the recognition of the peptide of SEQ. ID No: 2by cytolytic T cell clone CTL-IVSB but not cytolytic T cell clone CTL2/9.

FIG. 5 shows that the peptide of SEQ. ID No:2 is not recognized bycytolytic T cell clone CTL 210/9.

FIG. 6 shows the results obtained when TNF release assays were carriedout on various cells, including those which present HLA-B44 on theirsurface.

FIG. 7 shows, collectively, a series of chromium release assays usingpeptides described in this application on three different cell lines.

FIGS. 7A and 7D present experiments where the peptide of SEQ ID NO: 4was used.

FIGS. 7B and 7E show results where the peptide of SEQ ID NO: 5 was used.

FIGS. 7C and 7F set forth results obtained using SEQ ID NO: 2.

In FIG. 7, the symbol "∘" is used for cell line T2, " " for MZ2-MEL notpresenting HLA-A2, and " " for MZ2-MEL which has been transfected topresent HLA-A2. Example 12 elaborates on these tests.

FIGS. 8A and 8B show work using a cell line which presents MHC moleculeHLA-B44, and cytolytic T cell clone 22/31 ("CTL 22/31" hereafter). InFIG. 8A, the cell line ("Rosi EBV") was preincubated with monoclonalantibody W6/32, whereas in FIG. 8B, there was no preincubation.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS EXAMPLE 1

Melanoma cell lines SK 29-MEL (also referred to in the literature as SKMEL-29) and LB24-MEL, which have been available to researchers for manyyears, were used in the following experiments.

Samples containing mononuclear blood cells were taken from patients SK29(AV) and LB24 (these patients were also the source of SK 29-MEL andLB24-MEL, respectively). The melanoma cell lines were contacted to themononuclear blood cell containing samples. The mixtures were observedfor lysis of the melanoma cell lines, this lysis indicating thatcytolytic T cells ("CTLs") specific for a complex of peptide and HLAmolecule presented by the melanoma cells were present in the sample.

The lysis assay employed was a chromium release assay following Herin etal., Int. J. Cancer 39:390-396 (1987), the disclosure of which isincorporated by reference. The assay, however, is described herein. Thetarget melanoma cells were grown in vitro, and then resuspended at 10⁷cells/ml in DMEM, supplemented with 10 mM HEPES and 30% FCS, andincubated for 45 minutes at 37° C. with 200 μCi/ml of Na(⁵¹ Cr)O₄ .Labelled cells were washed three times with DMEM, supplemented with 10mM Hepes. These were then resuspended in DMEM supplemented with 10 mMHepes and 10 % FCS, after which 100 ul aliquots containing 10³ cells,were distributed into 96 well microplates. Samples of PBLs were added in100 ul of the same medium, and assays were carried out in duplicate.Plates were centrifuged for 4 minutes at 100 g, and incubated for fourhours at 37° C. in a 5.5 % of CO₂ atmosphere.

Plates were centrifuged again, and 100 ul aliquots of supernatant werecollected and counted. Percentage of ⁵¹ Cr release was calculated asfollows: ##EQU1## where ER is observed, experimental ⁵¹ Cr release, SRis spontaneous release measured by incubating 10³ labeled cells in 200ul of medium alone, and MR is maximum release, obtained by adding 100 ul0.3 % Triton X-100 to target cells.

Those mononuclear blood samples which showed high CTL activity wereexpanded and cloned via limiting dilution, and were screened again,using the same methodology.

The same method was used to test target K562 cells. When EBV-transformedB cells (EBV-B cells) were used, the only change was the replacement ofDMEM medium by Hank's medium, supplemented with 5 % FCS.

These experiments led to isolation of CTL clone "IVSB" from patient SK29(AV) and CTL clone 210/9 from patient LB24.

FIG. 1 presents the results of these assays, in panels A, B, G and I.Specifically, it will be seen that both CTLs lysed both melanoma celllines, and that there was no lysis of the K562 and EBV-B cell lines.

EXAMPLE 2

The CTLs described were tested against other melanoma cell lines todetermine whether their target was shared by other melanoma cell lines.Lysis as described in Example 1 was studied for lines LB4 .MEL, SK23.MEL (also known as SK MEL-23 ), and LE516 .MEL. FIG. 1, panels C, D andE shows that the clones did lyse these lines.

The tested lines are known to be of type HLA-A2, and the resultssuggested that the CTLs are specific for a complex of peptide and HLA-A2. This suggestion was verified by testing a variant of SK 29 -MEL whichhas lost HLA-A2 expression. FIG. 1, panel F shows these results. Neitherclone lysed the HLA-loss variant. When the variant was transfected withthe HLA-A2 gene of SK29 -MEL, however, and retested, lysis was observed.Thus, it can be concluded that the presenting molecule is HLA-A2.

EXAMPLE 3

Once the presenting HLA molecule was identified, studies were carriedout to identify the molecule, referred to hereafter as the "tumorrejection antigen precursor" or "TRAP" molecule which was the source ofthe presented peptide.

To do this, total RNA was isolated from cell line SK29 -MEL.1, which isa subclone of SK29 -MEL. The RNA was isolated using an oligo-dT bindingkit, following well recognized techniques. Once the total RNA wassecured, it was transcribed into cDNA, again using standardmethodologies. The cDNA was then ligated to EcoRI adaptors and clonedinto the EcoRI site of plasmid pcDNA-I/Amp, in accordance withmanufacturer's instructions. The recombinant plasmids were thenelectroporated into JM101 E. coli (electroporation conditions: 1 pulseat 25 μfarads, 2500 V).

The transfected bacteria were selected with ampicillin (50 μg/ml), andthen divided into 700 pools of 200 clones each. Each pool representedabout 100 different cDNAs, as analysis showed that about 50 % ofplasmids contained an insert. Each pool was amplified to saturation, andplasmid DNA was isolated via alkaline lysis, potassium acetateprecipitation and phenol extraction, following Maniatis et al., inMolecular Cloning: A Laboratory Manual (Cold Spring Harbor, N.Y., 1982). Cesium gradient centrifugation was not used.

EXAMPLE 4

The amplified plasmids were then transfected into eukaryotic cells.Samples of COS-7 cells were seeded, at 15,000 cells/well into tissueculture flat bottom microwells, in Dulbeco's modified Eagles Medium("DMEM") supplemented with 10 % fetal calf serum. The cells wereincubated overnight at 37° C., medium was removed and then replaced by30 μl/well of DMEM medium containing 10 % Nu serum, 400 μg/mlDEAE-dextran, 100 μM chloroquine, 100 ng of plasmid pcDNA-I/Amp-A2 and100 ng of DNA of a pool of the cDNA library described supra. PlasmidpcDNA-I/Amp-A2 contains the HLA-A2 gene from SK29 -MEL. Following fourhours of incubation at 37° C. the medium was removed, and replaced by 50μl of PBS containing 10 % DMSO. This medium was removed after twominutes and replaced by 200 μl of DMEM supplemented with 10 % of FCS.

Following this change in medium, COS cells were incubated for 48 hoursat 37° C. Medium was then discarded, and 2000 cells of either of thedescribed CTL clones were added, in 100μl of Iscove's medium containing10 % pooled human serum. When clone 210/9 was used, the medium wassupplemented with 25 U/ml of IL-2 . Supernatant was removed after 24hours, and TNF content was determined in an assay on WEHI cells, asdescribed by Traversari et al., Immunogenetics 35:145-152 (1992 ), thedisclosure of which is incorporated by reference.

Of 700 wells tested with IVSB, 696 showed between 0.6 and 4 pg of TNFper ml. The remaining four wells contained between 10 and 20 pg/ml ofTNF. Homologous wells tested with CTL 210/9 showed similar, clearlyhigher values. FIGS. 2 and 3 present these data.

EXAMPLE 5

Three of the four pools identified as high producers (numbers "123","181" and "384") were selected for further experiments. Specifically,the bacteria were cloned, and 570 bacteria were tested from each pool.Plasmid DNA was extracted therefrom, transfected into a new sample ofCOS cells in the same manner as described supra, and the cells wereagain tested for stimulation of CTL 210/9 and CTL IVSB. A positive clonewas found in pool 123 ("p123. B2"), and one was found in pool 384("p384.C6"). Convincing evidence that the transfected cells wererecognized by CTLs was obtained by carrying out a comparative test ofCOS cells transfected with cDNA and the HLA-A2 gene, and COS cellstransfected only with HLA-A2. TNF release in CTL supernatant wasmeasured by testing it on WEHI cells. The optical density of thesurviving WEHI cells was measured using MTT. Results are presented inTable 1 :

                  TABLE 1                                                         ______________________________________                                                 cDNA (123.B2) +                                                                           no cDNA +                                                         HLA-A2 DNA  HLA-A2                                                   ______________________________________                                        Run 1      0.087         0.502                                                Run 2      0.108         0.562                                                ______________________________________                                    

The values for WEHI OD's correspond to 24 pg/ml of TNF for cDNA andHLA-A2, versus 2.3 pg/ml for the control.

The plasmids from the positive clones were removed, and sequencedfollowing art known techniques. A sequence search revealed that theplasmid insert was nearly identical to the cDNA for human tyrosinase, asdescribed by Bouchard et al., J. Exp. Med. 169: 2029 (1989 ), thedisclosure of which is incorporated by reference. Thus, a normallyoccurring molecule (i.e., tyrosinase), may act as a tumor rejectionantigen precursor and be processed to form a peptide tumor rejectionantigen which is presented on the surface of a cell, in combination withHLA-A2, thereby stimulating lysis by CTL clones. The nucleic sequence ofthe identified molecule is presented as SEQ ID NO: 1.

EXAMPLE 6

Prior work reported by Chomez et al., Immunogenetics 35: 241 (1992 ) hasshown that small gene fragments which contain a sequence coding for anantigenic peptide resulted in expression of that peptide. This work,which is incorporated by reference in its entirety, suggested thecloning of small portions of the human tyrosinase cDNA described supraand in SEQ ID NO: 1 . Using the methodologies described in examples 1-5,various fragments of the cDNA were cotransfected with a gene for HLA-A2in COS-7 cells, and TNF release assays were performed. These experimentsled to identification of an approximately 400 base pair fragment which,when used in cotransfection experiments, provoked TNF release fromcytolytic T cell clone CTL IVSB discussed supra, shown to be specificfor HLA-A2 presenting cells. The 400 base fragment used corresponded tobases 711 to 1152 of SEQ ID NO: 1 . The amino acid sequence for whichthe fragment codes was deduced, and this sequence was then compared tothe information provided by Hunt et al., Science 255: 1261 (1992 ), andFalk et al., Nature 351: 290 (1991 ), the disclosures of which are bothincorporated by reference in their entirety. These references discussconsensus sequences for HLA-A2 presented peptides. Specifically, Huntdiscusses nonapeptides, where either Leu or Ile is always found at thesecond position, Leu being the "dominant residue". The ninth residue isdescribed as always being a residue with an aliphatic hydrocarbon sidechain. Val is the dominant residue at this position. Hunt discusses astrong signal for Leu and an intermediate signal for Met at the secondposition, one of Val, Leu, Ile or Thr at position 6, and Val or Leu atposition 9, with Val being particularly strong. On the basis of thecomparison, nonapeptides were synthesized and then tested to see if theycould sensitize HLA-A2 presenting cells. To do so, tyrosinase lossvariant cell lines SK29 -MEL 1.218 and T202 LB were used. Varyingconcentrations of the tested peptides were added to the cell lines,together with either of cytolytic T cell clone CTL IVSB or cytolytic Tcell clone CTL 2/9. Prior work, described supra, had established thatthe former clone lysed tyrosinase expressing cells which present HLA-A2,and that the latter did not.

The tyrosinase loss variants were incubated for one hour in a solutioncontaining ⁵¹ Cr, at 37° C., either with or without anti HLA-A2 antibodyMA2.1, which was used to stabilize empty HLA-A2 molecules. In the tests,cells were washed four times, and then incubated with varying dilutionsof the peptides, from 100 μM down to 0.01 μM. After 30 minutes, effectorcells were added at an E/T ratio of 40/1 and four hours later, 100λ ofsupernatant were collected and radioactivity counted.

FIG. 4 shows the results obtained with nonapeptide

    Tyr Met Asn Gly Thr Met Ser Gln Val.                       (SEQ ID NO: 2 ).

This peptide, referred to hereafter as SEQ ID NO: 2, corresponds toresidues 1129-1155 of the cDNA sequence for tyrosinase presented in SEQID NO: 1 . Complexes of HLA-A2 and this peptide are recognized by CTLclone IVSB.

In a parallel experiment, it was shown that CTL clone CTL 210/9, derivedfrom patient LB24, did not recognize the complexes of HLA-A2 and thepeptide of SEQ ID NO: 2, although it did recognize complexes of HLA-A2and a tyrosinase derived peptide. Thus, tyrosinase is processed to atleast one additional peptide which, when presented by HLA-A2 molecules,is recognized by CTL clones.

EXAMPLE 7

In a follow-up experiment, a second gene fragment which did not encodethe peptide of SEQ ID NO: 2 was used. This fragment began at base 1 andended at base 1101 of SEQ ID NO: 1 (i.e. the EcoRI-SphI fragment).Cytolytic T cell clone CTL 210/9, discussed supra, was tested againstCOS-7 cells transfected with this fragment in the manner describedsupra. CTL IVSB was also tested. These results, showed that CTL 210/9recognized an antigen on the surface of HLA-A2 expressing cellstransfected with this fragment, but CTL IVSB did not. Thus, a secondtumor rejection antigen peptide is derived from tyrosinase.

EXAMPLE 8

In order to further define the tumor rejection antigen recognized by CTL210/9, the following experiments were carried out.

A second fragment, corresponding to bases 451-1158 of SEQ ID NO: 1 wastransfected into COS cells together with a gene for HLA-A2, and TNFrelease assays were carried out. This sequence provoked TNF release fromclone IVSB (20 pg/ml), but not from LB24 -CTL 210/9 (3.8 pg/ml). Theseresults confirmed that the two CTL clones recognize different peptides,and that the peptide recognized by CTL 210/9 must be encoded by region1-451.

EXAMPLE 9

The tyrosinase derived peptide coded for by cDNA fragment 1-451 wasanalyzed for consensus sequences known to bind HLA-A2 . The peptidescorresponding to these consensus sequences were synthesized, and testedfor their ability to sensitize HLA-A2 presenting cells. To do so, twotyrosinase negative melanoma cell lines were used (i.e., NA8 -MEL, andMZ2 -MEL 2.2 transfected with HLA-A2 ), and cell line T2, as describedby Salter et al, Immunogenetics 21: 235-246 (1985 )).

The cells were incubated with ⁵¹ Cr, and monoclonal antibody MA.2.1,which is specific for HLA-A2 for 50 minutes at 37° C., followed bywashing (see Bodmer et al., Nature 342: 443-446 (1989 ), the disclosureof which is incorporated by reference in its entirety). Target cellswere incubated with various concentrations of the peptides, and witheither of LB 24 -CTL clones 210/5 or 210/9 . The percent of chromiumrelease was measured after four hours of incubation.

The peptide Met Leu Leu Ala Val Leu Tyr Cys Leu Leu (SEQ ID NO: 3 ) wasfound to be active.

In further experiments summarized here, CTL-IVSB previously shown torecognize SEQ ID NO:2 , did not recognize the peptide of SEQ ID NO: 3.

The results are summarized in Tables 2-4 which follow:

                  TABLE 2                                                         ______________________________________                                                  Peptide                                                                       SEQ ID NO: 2                                                                            SEQ ID NO: 3                                              ______________________________________                                        CTL-IVSB    +           -                                                     CTL-210/5   -           +                                                     CTL-210/9   -           +                                                     ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        Lysis of MZ2-2.2.2 sensitized with tyrosinase                                 peptides by CTL 210/5, CTL 210/9, and CTL IVSB                                                                MZ2.22.A2 +                                   Effectors  Peptides    Dose     anti-A2*                                      ______________________________________                                        CTL 210/5  SEQ ID NO: 3                                                                              10    μM                                                                              13                                                                 1          17                                          (44:1)     SEQ ID NO: 2                                                                              30    μM                                                                              16                                                                 10         1                                                                  3          1                                                                             1                                                                  10    μm                                                                              13                                                                 3          17                                          CTL 210/9  SEQ ID NO: 3                                                                              1          15                                          (20:1)     SEQ ID NO: 2                                                                              30    M    1                                                                  10         1                                                                  3          1                                                                  10    μM                                                                              1                                           CTL IVSB   SEQ ID NO: 3                                                                              3          1                                           (40:1)                 1          1                                                      SEQ ID NO: 2                                                                              30    μM                                                                              63                                                                 10         63                                                                 3          62                                          ______________________________________                                         *Target cells were incubated with .sup.51 Cr and monoAb MA2.1 (antiHLA-A2     for 50 minutes, then washed three times. They were incubated with various     concentration of peptides for 30 min. CTL cells were added at the             indicated (E:T) ratio. The % specific .sup.51 Cr release was measured         after 4 h incubation.                                                    

                                      TABLE 4                                     __________________________________________________________________________    Test of tyrosinase peptides recognized by LB24-CTL 210/5 and 210/9            or SK29-CTL IVSB                                                                                   (% Cr51 specific release)                                Effectors                                                                           Peptides Dose  NAS-MEL                                                                             MZ2-2.2:A2                                                                           T2                                          __________________________________________________________________________    LB24-CTL                                                                            MLLAVLYCLL                                                                             10 μm                                                                            30    31     36                                          210/5 (LAUS 17-5)                                                                            3     23    27     35                                          (41:1)         1     17    20     26                                                         300                                                                              nM 6     17     16                                                         100   2     8      5                                                          30    3     5      2                                                          0     0     0      0                                           LB24-CTL                                                                            MLLAVLYCLL                                                                             10 μM                                                                            14    19     21                                          210/9 (LAUS 17-5)                                                                            3     13    17     20                                          (28:1)         1     9     14     13                                                         300                                                                              nM 3     9      5                                                          100   1     1      1                                                          30    0     1      0                                                          0     0     1      0                                           SK29-CTL                                                                            YMNGTMSQV                                                                              10 μM                                                                            46    46     59                                          IVSB  (MAINZ)  3     38    44     52                                          (42:1)         1     27    40     46                                                         300                                                                              nM 14    22     34                                                         30    1     9      10                                                         10    1     3      3                                                          3     0     3      4                                                          1     0     1      0                                                          0     0     4      0                                           spt rel.             339   259    198                                         max-spt              2594  1693   1206                                        %                    11    13     14                                          __________________________________________________________________________

EXAMPLE 10

Additional experiments were carried out using CTL clone 22/31. Thisclone had previously been shown to lyse subline MZ2 -MEL.43 fromautologous melanoma cell line MZ2 -MEL, but did not lyse other sublines,such as MZ2 -MEL 3.0 and MZ2 -MEL 61.2, nor did it lyse autologous EBVtransformed B cells, or killer cell line K562 (see Van den Eynde et al.,Int. J. Cancer 44: 634-640 (1989 )). The antigen presented by MZ2-MEL.43 is referred to as antigen C.

In prior work including that reported in the parent of this application,it was found that the tyrosinase gene encodes an antigen recognized byautologous CTLs on most HLA-A2 expressing melanomas. Expression of thisgene in sublines of cell line MZ2 -MEL was tested by PCR amplification.Clone MZ2 -MEL.43 was found to be positive, whereas other MZ2 -MELclones, such as MZ2 -MEL.3.0 were negative. Correlation of expression ofthe tyrosinase gene, and antigen MZ2 -C, suggested that MZ2 -C might bea tumor rejection antigen derived from tyrosinase, and presented by anHLA molecule expressed by MZ2 -MEL. This cell line does not expressHLA-A2, which would indicate that if a tyrosinase derived peptide werepresented as a TRA, a second HLA molecule was implicated.

Studies were carried out to identify which HLA molecule presentedantigen C to CTL 22/31. To determine this, cDNA clones of the HLAmolecules known to be on the cell surface, i.e., HLA-A29, HLA-B37, HLA-B44.02, and HLA-C clone 10, were isolated from an MZ2 -MEL.43 cDNAlibrary, and then cloned into expression vector pcDNAI/Amp. RecipientCOS 7 cells were then transfected with one of these constructs or aconstruct containing HLA-A1, plus cDNA coding for tyrosinase (SEQ ID NO:1 ). The contransfection followed the method set forth above. One daylater CTL 22/31 was added, and 24 hours later, TNF release was measuredby testing cytotoxicity on WEHI-164 -13, following Traversari et al,supra. FIG. 6 shows that TNF was released by CTL 22/31 only in thepresence of cells transfected with both HLA-B44 and tyrosinase. Theconclusion to be drawn from this is that HLA-B44 presents a tyrosinasederived tumor rejection antigen.

EXAMPLE 11

The experiments described supra showed, inter alia, that the decamer ofSEQ ID NO:3 effectively induced lysis of HLA-A2 presenting cells. It isfairly well accepted that MHC molecules present nonapeptides. To thatend, experiments were carried out wherein two nonamers were tested,which were based upon the decapeptide which did give positive results.Specifically, either the first or tenth amino acid was omitted to createtwo peptides, i.e.:

    Met Leu Leu Ala Val Leu Tyr Cys Leu                        (SEQ ID NO: 4 )

    Leu Leu Ala Val Leu Tyr Cys Leu Leu                        (SEQ ID NO: 5 ).

These peptides were tested in the same way the decapeptide was tested,as set forth in the prior examples at concentrations ranging from 10 μMto 1 nM. Three presenting cells were used. As summarized in Table 5,which follows, "T2" is a mutant human cell line, "CEMX721.174 T2" asdescribed by Salter, Immunogenetics 21: 235 (1985 ). This line presentsHLA-A2. "G2.2" is a variant of the cell line MZ2 -MEL. The variant hasbeen transfected with a gene coding for HLA-A2. The abbreviation"G2.2.5" stands for a variant which does not express HLA-A2. All cellswere incubated with monoclonal antibody MA2.1 prior to contact with thecytolytic T cell clone. This procedure stabilizes so-called "empty" MHCmolecules, although the mechanism by which this occurs is not wellunderstood and effector CTLs 210/5 and 210/9 were both used. At aconcentration of 10 μM, the nonamer of SEQ ID NO: 4 was twice aseffective when used with CTL clone 210/5, and four times as effectivewith clone 210/9 whereas the nonamer of SEQ ID NO: 5 was ineffective atinducing lysis.

EXAMPLE 12

In further experiments, chromium release assays were carried out usingthe peptides of SEQ ID NOS: 4 and 5, as well as SEQ ID NO: 2. The targetcells were allogeneic melanoma cells, i.e., MZ2 -MEL, previouslytransfected with HLA-A2, and cell line T2, which presents HLA-A2, buthas an antigen processing defect which results in an increased capacityto present exogenous peptides (Cerundolo et al., Nature 345: 449 (1990)). All cells were pretreated with monoclonal antibody MA2.1 for fiftyminutes. The cells were incubated with the peptide of choice, for 30minutes, at various concentrations. Then, one of CTL clones 210/9 andISVB was added in an effector: target ratio of 60. Chromium release wasmeasured after four hours, in the manner described supra.

The results are presented in FIG. 7, i.e., FIGS. 7A-7C. The peptide ofSEQ ID NO: 4 sensitized cells to CTL 210/9, while SEQ ID NO: 5 did not.SEQ ID NO: 6 sensitized cells to CTL IVSB, as already noted in previousexamples.

EXAMPLE 13

Work which followed up on the experiments set forth in example 10 wasthen carried out, in an effort to define the antigenic peptide presentedby HLA-B44. To do so, cDNA sequences corresponding to fragments of thetyrosinase cDNA sequence were cotransfected, together with a gene codingfor HLA-B44, into COS-7 cells. The protocol is essentially thatdescribed in example 6, supra. The cytolytic T cell clone 22/31,discussed supra, was used. TNF release was determined. Two fragments,i.e., base fragments 1-611, and 427-1134 induced TNF release. Thissuggested that the presented peptide was in the overlapping region. As aresult of this observation, shorter fragments were tested. Fragmentsbeginning at positions 385, 442, 514 and 574 were able to induce TNFrelease, while fragments starting at positions 579 and 585 were not.These observations, in turn, suggested the synthesis, following standardmethodologies, of a 13 amino acid peptide beginning at position 574.

This peptide was then used in experiments to determine whether itinduced lysis by CTL 22/31. Table 5, which follows, shows that the 13-mer rendered two EBV transfected cell lines which express HLA-B44sensitive to lysis.

                  TABLE 5                                                         ______________________________________                                        10F94-tyros  13-mer  sur  EBV-I                                               1          2             3         4                                          Effector   Dose pept 13 A.A.                                                                           Rosi-EBV  MZ2-EBV                                    ______________________________________                                        1     MZ2-CTL-22/31 Ser Glu Ile Trp Arg Asp Ile Asp Phe                             Ala His Glu Ala (SEQ ID NO: 6)                                          3     60:1     30     μM    83      71                                     4              10              85      72                                     5              3               77      66                                     6              1               79      63                                     7              300    nM       60      33                                     8              100             44      17                                     9              30              21      4                                      10             10              9       5                                      11             3               10      6                                      12                                                                            13             0               10      6                                      14                                                                            15    spt.rel.                 393     472                                    16    max.rel.                 1698    1792                                   17    %                        23      26                                     ______________________________________                                    

As a follow up, even shorter peptides were tested. A decamercorresponding to nucleotide bases 574-604, i.e.

    Ser Glu Ile Trp Arg Asp Ile Asp Phe Ala                    (SEQ ID NO: 7 )

did provoke lysis, as did peptide:

    Ser Glu Ile Trp Arg Asp Ile Asp Phe                        (SEQ ID NO: 8 )

The nonamer:

    Glu Ile Trp Arg Asp Ile Asp Phe Ala                        (SEQ ID NO: 9 )

in contrast, was not recognized. Table 6, Which follows, summarizesthese results, which are also depicted in FIG. 8.

The only other peptide reported to be bound by HLA-B44 is

    Glu Glu Asn Leu Leu Asp Phe Val Arg Phe                    (SEQ ID NO: 10 )

as reported by Burrows et al., J. Virol 64: 3974 (1990 ). The datadescribed supra suggest that Glu at second position and Phe in ninthposition may represent anchor residues for HLA-B44.

                                      TABLE 6                                     __________________________________________________________________________                     3     4     5     6     7     8                              1          2     +W SEQ                                                                              +W SEQ                                                                              +W SEQ                                                                              -W SEQ                                                                              -W SEQ                                                                              -W SEQ                         Effector   Dose  ID NO: 7                                                                            ID NO: 8                                                                            ID NO: 9                                                                            ID NO: 7                                                                            ID NO: 8                                                                            ID NO: 9                       __________________________________________________________________________    1 MZ2-CTL-22/31                                                                          1  μM                                                                            91    93    7     98    99    11                             2          300                                                                              nM 76    81    4     77    97    6                              3 45:1     100   43    73    2     45    64    8                              4          30    17    37    0     15    21    6                              5          10    4     12    1     5     8     4                              6          3     3     4     1     0     7     2                              7          1     2     4                 3                                    8          0.3   1     1                 2                                    __________________________________________________________________________

The foregoing experiments demonstrate that tyrosinase is processed as atumor rejection antigen precursor, leading to formation of complexes ofthe resulting tumor rejection antigens with a molecule on at least someabnormal cells, for example, melanoma cells with HLA-A2 or HLA-B44phenotype. The complex can be recognized by CTLs, and the presentingcell lysed. This observation has therapeutic and diagnosticramifications which are features of the invention. With respect totherapies, the observation that CTLs which are specific for abnormalcells presenting the aforementioned complexes are produced, suggestsvarious therapeutic approaches. One such approach is the administrationof CTLs specific to the complex to a subject with abnormal cells of thephenotype at issue. It is within the skill of the artisan to developsuch CTLs in Vitro. Specifically, a sample of cells, such as bloodcells, are contacted to a cell presenting the complex and are capable ofprovoking a specific CTL to proliferate. The target cell can be atransfectant, such as a COS cell of the type described supra. Thesetransfectants present the desired complex on their surface and, whencombined with a CTL of interest, stimulate its proliferation. So as toenable the artisan to produce these CTLs, vectors containing the genesof interest, i.e., pcDNA-1/ Amp1 (HLA-A2 ), and p123. B2 (humantyrosinase), have been deposited in accordance with the Budapest Treatyat the Institut Pasteur, under Accession Numbers I1275 and I1276,respectively. COS cells, such as those used herein are widely available,as are other suitable host cells.

To detail the therapeutic methodology, referred to as adoptive transfer(Greenberg, J. Immunol. 136 (5 ): 1917 (1986 ); Reddel et al., Science257: 238 (Jul. 10, 1992 ); Lynch et al., Eur. J. Immunol. 21: 1403-1410(1991 ); Kast et al., Cell 59: 603-614 (Nov. 17, 1989 )), cellspresenting the desired complex are combined with CTLs leading toproliferation of the CTLs specific thereto. The proliferated CTLs arethen administered to a subject with a cellular abnormality which ischaracterized by certain of the abnormal cells presenting the particularcomplex. The CTLs then lyse the abnormal cells, thereby achieving thedesired therapeutic goal.

The foregoing therapy assumes that at least some of the subject'sabnormal cells present one or more of the HLA/tyrosinase derived peptidecomplexes. This can be determined very easily. For example CTLs areidentified using the transfectants discussed supra, and once isolated,can be used with a sample of a subject's abnormal cells to determinelysis in vitro. If lysis is observed, then the use of specific CTLs insuch a therapy may alleviate the condition associated with the abnormalcells. A less involved methodology examines the abnormal cells for theirHLA phenotype, using standard assays, and determines expression oftyrosinase via amplification using, e.g., PCR. The fact that a pluralityof different HLA molecules present TRAs derived from tyrosinaseincreases the number of individuals who are suitable subjects for thetherapies discussed herein.

Adoptive transfer is not the only form of therapy that is available inaccordance with the invention. CTLs can also be provoked in vivo, usinga number of approaches. One approach, i.e., the use of non-proliferativecells expressing the complex, has been elaborated upon supra. The cellsused in this approach may be those that normally express the complex,such as irradiated melanoma cells or cells transfected with one or bothof the genes necessary for presentation of the complex. Chen et al.,Proc. Natl. Acad. Sci. USA 88: 110-114 (January, 1991 ) exemplifies thisapproach, showing the use of transfected cells expressing HPVE7 peptidesin a therapeutic regime. Various cell types may be used. Similarly,vectors carrying one or both of the genes of interest may be used. Viralor bacterial vectors are especially preferred. In these systems, thegene of interest is carried by, e.g., a Vaccinia virus or the bacteriaBCG, and the materials de facto "infect" host cells. The cells whichresult present the complex of interest, and are recognized by autologousCTLs, which then proliferate. A similar effect can be achieved bycombining tyrosinase itself with an adjuvant to facilitate incorporationinto HLA-A2 presenting cells. The enzyme is then processed to yield thepeptide partner of the HLA molecule.

The foregoing discussion refers to "abnormal cells" and "cellularabnormalities". These terms are employed in their broadestinterpretation, and refer to any situation where the cells in questionexhibit at least one property which indicates that they differ fromnormal cells of their specific type. Examples of abnormal propertiesinclude morphological and biochemical changes, e.g. Cellularabnormalities include tumors, such as melanoma, autoimmune disorders,and so forth.

The invention also provides a method for identifying precursors to CTLtargets. These precursors are referred to as tumor rejection antigenswhen the target cells are tumors, but it must be pointed out that whenthe cell characterized by abnormality is not a tumor, it would besomewhat misleading to refer to the molecule as a tumor rejectionantigen. Essentially, the method involves identifying a cell which isthe target of a cytolytic T cell of the type discussed supra. Once sucha cell is identified, total RNA is converted to a cDNA library, which isthen transfected into a cell sample capable of presenting an antigenwhich forms a complex with a relevant HLA molecule. The transfectantsare contacted with the CTL discussed supra, and again, targeting by theCTL is observed (lysis and/or TNF production). These transfectants whichare lysed are then treated to have the cDNA removed and sequenced, andin this manner a precursor for an abnormal condition, such as a tumorrejection antigen precursor, can be identified.

Other aspects of the invention will be clear to the skilled artisan andneed not be repeated here.

The terms and expressions which have been employed are used as terms ofdescription and not of limitation, and there is no intention in the useof such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, it being recognizedthat various modifications are possible within the scope of theinvention.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 10                                                 (2) INFORMATION FOR SEQ ID NO: 1:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1894 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:                                      GGAAGAAT GCTCCTGGCTGTTTTGTACTGCCTGCTGTGGAGTTTCCAG48                           GlyArgMetLeuLeuAlaValLeuTyrCysLeuLeuTrpSerPheGln                              -15-10-5                                                                      ACCTCC GCTGGCCATTTCCCTAGAGCCTGTGTCTCCTCTAAGAACCTG96                           ThrSerAlaGlyHisPheProArgAlaCysValSerSerLysAsnLeu                              1510                                                                          ATGGAGAAGGAA TGCTGTCCACCGTGGAGCGGGGACAGGAGTCCCTGT144                          MetGlyLysGluCysCysProProTrpSerGlyAspArgSerProCys                              15202530                                                                      GGCCAG CTTTCAGGCAGAGGTTCCTGTCAGAATATCCTTCTGTCCAAT192                          GlyGlnLeuSerGlyArgGlySerCysGlnAsnIleLeuLeuSerAsn                              354045                                                                        GCA CCACTTGGGCCTCAATTTCCCTTCACAGGGGTGGATGACCGGGAG240                          AlaProLeuGlyProGlnPheProPheThrGlyValAspAspArgGlu                              505560                                                                        TCG TGGCCTTCCGTCTTTTATAATAGGACCTGCCAGTGCTCTGGCAAC288                          SerTrpProSerValPheTyrAsnArgThrCysGlnCysSerGlyAsn                              657075                                                                        TTCATG GGATTCAACTGTGGAAACTGCAAGTTTGGCTTTTGGGGACCA336                          PheMetGlyPheAsnCysGlyAsnCysLysPheGlyPheTrpGlyPro                              808590                                                                        AACTGCACAGAG AGACGACTCTTGGTGAGAAGAAACATCTTCGATTTG384                          AsnCysThrGluArgArgLeuLeuValArgArgAsnIlePheAspLeu                              95100105110                                                                   AGTGCC CCAGAGAAGGACAAATTTTTTGCCTACCTCACTTTAGCAAAG432                          SerAlaProGluLysAspLysPhePheAlaTyrLeuThrLeuAlaLys                              115120125                                                                     CAT ACCATCAGCTCAGACTATGTCATCCCCATAGGGACCTATGGCCAA480                          HisThrIleSerSerAspTyrValIleProIleGlyThrTyrGlyGln                              130135140                                                                     ATG AAAAATGGATCAACACCCATGTTTAACGACATCAATATTTATGAC528                          MetLysAsnGlySerThrProMetPheAsnAspIleAsnIleTyrAsp                              145150155                                                                     CTCTTT GTCTGGATGCATTATTATGTGTCAATGGATGCACTGCTTGGG576                          LeuPheValTrpIleHisTyrTyrValSerMetAspAlaLeuLeuGly                              160165170                                                                     GGATCTGAAATC TGGAGAGACATTGATTTTGCCCATGAAGCACCAGCT624                          GlyTyrGluIleTrpArgAspIleAspPheAlaHisGluAlaProAla                              175180185190                                                                  TTTCTG CCTTGGCATAGACTCTTCTTGTTGCGGTGGGAACAAGAAATC672                          PheLeuProTrpHisArgLeuPheLeuLeuArgTrpGluGlnGlyIle                              195200205                                                                     CAG AAGCTGACAGGAGATGAAAACTTCACTATTCCATATTGGGACTGG720                          GlnLysLeuThrGlyAspGlyAsnPheThrIleProTyrTrpAspTrp                              210215220                                                                     CGG GATGCAGAAAAGTGTGACATTTGCACAGATGAGTACATGGGAGGT768                          ArgAspAlaGluLysCysAspIleCysThrAspGlyTyrMetGlyGly                              225230235                                                                     CAGCAC CCCACAAATCCTAACTTACTCAGCCCAGCATCATTCTTCTCC816                          GlnHisProThrAsnProAsnLeuLeuSerProAlaSerPhePheSer                              240245250                                                                     TCTTGGCAGATT GTCTGTAGCCGATTGGAGGAGTACAACAGCCATCAG864                          SerTrpGlnIleValCysSerArgLeuGluGluTyrAsnSerHisGln                              255260265270                                                                  TCTTTA TGCAATGGAACGCCCGAGGGACCTTTACGGCGTAATCCTGGA912                          SerLeuCysAsnGlyThrProGluGlyProLeuArgArgAsnProGly                              275280285                                                                     AAC CATGACAAATCCAGAACCCCAAGGCTCCCCTCTTCAGCTGATGTA960                          AsnHisAspLysSerArgThrProArgLeuProSerSerAlaAspVal                              290295300                                                                     GAA TTTTGCCTGAGTTTGACCCAATATGAATCTGGTTCCATGGATAAA1008                         GluPheCysLeuSerLeuThrGlnTyrGluSerGlySerMetAspLys                              305310315                                                                     GCTGCC AATTTCAGCTTTAGAAATACACTGGAAGGATTTGCTAGTCCA1056                         AlaAlaAsnPheSerPheArgAsnThrLeuGluGlyPheAlsSerPro                              320325330                                                                     CTTACTGGGATA GCGGATGCCTCTCAAAGCAGCATGCACAATGCCTTG1104                         LeuThrGlyIleAlaAspAlaSerGlnSerSerMetHisAsnAlaLeu                              335340345350                                                                  CACATC TATATGAATGGAACAATGTCCCAGGTACAGGGATCTGCCAAC1152                         HisIleTyrMetAsnGlyThrMetSerGlnMetGlnGlySerAlaAsn                              355360365                                                                     GAT CCTATCTTCCTTCTTCACCATGCATTTGTTGACAGTATTTTTGAG1200                         AspProIlePheLeuLeuHisHisAlaPheValAspSerIlePheGlu                              370375380                                                                     CAG TGGCTCCAAAGGCACCGTCCTCTTCAAGAAGTTTATCCAGAAGCC1248                         GlnTrpLeuArgArgHisArgProLeuGlnGluValTyrProGluAla                              385390395                                                                     AATGCA CCCATTGGACATAACCGGGAATCCTACATGGTTCCTTTTATA1296                         AsnAlaProIleGlyHisAsnArgGluSerTyrMetValProPheIle                              400405410                                                                     CCACTGTACAGA AATGGTGATTTCTTTATTTCATCCAAAGATCTGGGC1344                         ProLeuTyrArgAsnGlyAspPhePheIleSerSerLysAspLeuGly                              415420425430                                                                  TATGAC TATAGCTATCTACAAGATTCAGACCCAGACTCTTTTCAAGAC1392                         TyrAspTyrSerTyrLeuGlnAspSerAspProAspSerPheGlnAsp                              435440445                                                                     TAC ATTAAGTCCTATTTGGAACAAGCGAGTCGGATCTGGTCATGGCTC1440                         TyrIleLysSerTyrLeuGlyGlnAlaSerArgIleTrpSerTrpLeu                              450455460                                                                     CTT GGGGCGGCGATGGTAGGGGCCGTCCTCACTGCCCTGCTGGCAGGG1488                         LeuGlyAlaAlaMetValGlyAlaValLeuThrAlaLeuLeuAlaGly                              465470475                                                                     CTTGTG AGCTTGCTGTGTCGTCACAAGAGAAAGCAGCTTCCTGAAGAA1536                         LeuValSerLeuLeuCysArgHisLysArgLysGlnLeuProGluGlu                              480485490                                                                     AAGCAGCCACTC CTCATGGAGAAAGAGGATTACCACAGCTTGTATCAG1584                         LysGlnProLeuLeuMetGluLysGluAspTyrHisSerLeuTyrGln                              495500505510                                                                  AGCCAT TTA1593                                                                SerHisLeu                                                                     513                                                                           TAAAAGGCTTAGGCAATAGAGTAGGGCCAAAAAGCCTGACCTCACTCTAACTCAAAGTAA1653              TGTCCAGGTTCCCAGAGAATATCTGC TGGTATTTTTCTGTAAAGACCATTTGCAAAATTG1713             TAACCTAATACAAAGTGTAGCCTTCTTCCAACTCAGGTAGAACACACCTGTCTTTGTCTT1773              GCTGTTTTCACTCAGCCCTTTTAACATTTTCCCCTAAGCCCATATGTCTAAGGAAAGGAT1833               GCTATTTGGTAATGAGGAACTGTTATTTGTATGTGAATTAAAGTGCTCTTATTTTAAAAA1893             A1894                                                                         (2) INFORMATION FOR SEQ ID NO: 2:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acid residues                                              (B) TYPE: amino acid                                                         (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:                                      TyrMetAsnGlyThrMetSerGlnVal                                                   (2) INFORMATION FOR SEQ ID NO: 3:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acid residues                                             (B) TYPE: amino acid                                                         (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:                                      MetLeuLeuAlaValLeuTyrCysLeuLeu                                                510                                                                           (2) INFORMATION FOR SEQ ID NO: 4:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 9 amino acid residues                                            (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:                                      MetLeuLeuAlaValLeuTyrCysLeu                                                   5                                                                             (2) INFORMATION FOR SEQ ID NO: 5:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acid residues                                             (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:                                      LeuLeuAlaValLeuTyrCysLeuLeu                                                   5                                                                             (2) INFORMATION FOR SEQ ID NO: 6:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino acid residues                                             (B) TYPE: amino acid                                                         (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:                                      SerGluIleTrpArgAspIleAspPheAlaHisGluAla                                       510                                                                           (2) INFORMATION FOR SEQ ID NO: 7:                                              (i) SEQUENCE CHARACTERISTICS:                                                (A) LENGTH: 10 amino acid residues                                            (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:                                      SerGluIleTrpArgAspIleAspPheAla                                                510                                                                           (2) INFORMATION FOR SEQ ID NO: 8:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acid residues                                             (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:                                      SerGluIleTrpArgAspIleAspPhe                                                   5                                                                             (2 ) INFORMATION FOR SEQ ID NO: 9:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acid residues                                             (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:                                      GluIleTrpArgAspIleAspPheAla                                                   5                                                                             (2) INFORMATION FOR SEQ ID NO: 10:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acid residues                                            (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:                                     GluGluAsnLeuLeuAspPheValArgPhe                                                5 10                                                                      

We claim:
 1. Isolated peptide of SEQ ID NO:
 7. 2. Isolated peptide ofSEQ ID NO: 8.